Ethanolic extracts of babandotan leaves (Ageratum conyzoides L ) prevents inflammation and proteoglycan degradation by inhibiting TNF α and MMP 9 on osteoarthritis rats induced by monosodium iodoaceta[.]
Accepted Manuscript Ethanolic extracts of babandotan leaves (Ageratum conyzoides L.) prevents inflammation and proteoglycan degradation by inhibiting TNF-α and MMP-9 on osteoarthritis rats induced by monosodium iodoacetate Anton Bahtiar, Mutiara Nurazizah, Tirza Roselina, Anita Paulina Tambunan, Ade Arsianti PII: S1995-7645(16)30443-6 DOI: 10.1016/j.apjtm.2017.03.006 Reference: APJTM 422 To appear in: Asian Pacific Journal of Tropical Medicine Received Date: 10 November 2016 Revised Date: 10 January 2017 Accepted Date: 15 January 2017 Please cite this article as: Bahtiar A, Nurazizah M, Roselina T, Tambunan AP, Arsianti A, Ethanolic extracts of babandotan leaves (Ageratum conyzoides L.) prevents inflammation and proteoglycan degradation by inhibiting TNF-α and MMP-9 on osteoarthritis rats induced by monosodium iodoacetate, Asian Pacific Journal of Tropical Medicine (2017), doi: 10.1016/j.apjtm.2017.03.006 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Normal Knee Joint RI PT Stimulus Eg Monosodium Iodoacetate M AN U SC Osteoarthritis of Knee Joint TE D TNFα MMP’s Eg MMP9 AC C EP Babandotan extract Inflammation Proteoglycan degradation ACCEPTED MANUSCRIPT Ethanolic Extracts of Babandotan Leaves (Ageratum conyzoides L.) prevents inflammation and proteoglycan degradation by inhibiting TNFα and MMP9 on Osteoarthritis Rat Induced by Monosodium Iodoacetate Anton Bahtiar1*, Mutiara Nurazizah1, Tirza Roselina1, Anita Paulina Tambunan1, Ade Arsianti2 RI PT Faculty of Pharmacy, Universitas Indonesia, Kampus UI Depok Indonesia 16424 Faculty of Medicine, Universitas Indonesia, Kampus UI Salemba Raya no 6, Indonesia * Corresponding author : Anton Bahtiar, Faculty of Pharmacy, Universitas Indonesia, kampus baru UI, Depok Indonesia 16424 Tel : +62-21-7864049; Fax : +62-21-7863433 E-mail address : anton.bahtiar@ui.ac.id AC C EP TE D M AN U SC ACCEPTED MANUSCRIPT Ethanolic extracts of babandotan leaves (Ageratum conyzoides L.) prevents inflammation and proteoglycan degradation by inhibiting TNF-α and MMP-9 on osteoarthritis rats induced by monosodium RI PT iodoacetate Anton Bahtiar1, Mutiara Nurazizah1, Tirza Roselina1, Anita Paulina Tambunan1, Ade Arsianti2 Faculty of Pharmacy, Universitas Indonesia, Kampus UI, Depok 16424, Indonesia Faculty of Medicine, Universitas Indonesia, Kampus UI Salemba Raya no 6, Indonesia SC Keywords: M AN U Inflammation Osteoarthritis Ageratum conyzoides Hematology parameters TNF-α MMP-9 corresponding author: Anton Bahtiar, Faculty of Pharmacy, Universitas Indonesia, kampus TE D First and baru UI, Depok 16424, Indonesia Tel: +62-21-7864049 Fax: +62-21-7863433 E-mail: anton.bahtiar@ui.ac.id EP Foundation project: This work was supported by the Ministry of Research Technology and Higher Education of Indonesia 2016 AC C Article history: Received 10 November 2016 Received in revised form 10 January 2017 Accepted 15 January 2017 Available online 20 March 2017 This paper has Figures and Tables ABSTRACT Objective: To analyze the effects of Ageratum conyzoides L on the monosodium iodoacetate induced osteoarthritis rats Methods: Thin layer chromatography was performed to analyze the constituents of the babandotan extract leaves White male Sprague-Dawley rats used in this study were divided into groups: normal control and negative control groups, both given 0.5% carboxymethyl ACCEPTED MANUSCRIPT cellulose; the positive control group that was given glucosamine and chondroitin suspension (486 mg/200 g B.W.); the dose variation extract groups including dose 1, 2, and that were given 40, 80, and 160 mg/200 g B.W respectively on day 29 until 50 All the groups were induced with 0.05 mL monosodium iodoacetate (20 mg/mL) on day 1, except normal control RI PT induced by saline Measurement of edema volume of rat knees was performed on day 0, 8, 15, 22, 29, 43, and 50 Hematology data was measured at day 1, 29 and 50 Serum was collected at day 50 to evaluate TNF-α and MMP-9 by ELISA Cartilage histopathology was evaluated by staining with H&E and Safranin-o-fast green staining on day 50 Results: The babandotan leaves extract dose (80 mg/200 g B.W.) and dose (160 mg/200 g SC B.W.) could decrease the edema volume, increase the area and thickness of articular cartilage, and increase proteoglycan level Particularly, dose (160 mg/200 g B.W.) of M AN U extract babandotan leaves were able to significantly decrease the number of leukocytes, lymphocytes and udem volume, and decrease TNF alpha and MMP-9 levels Conclusions: Babandotan leaves extract can recover inflammation and cartilages degradation by inhibiting TNF-α in inflammation processes and MMP-9 in the collagenase reaction in the Introduction TE D cartilages Osteoarthritis (OA) is characterized by joint pain, limited motion, and inflammation without systemic effects[1] According to the World Health Organization (WHO) data, OA is EP one of the main causes of malfunctions and reduces the quality of 151 million people’s life worldwide and 24 million people’s in Southeast Asia[2] Indonesia ranked the 4th with AC C highest number of elderly people affected with OA after China, India and the United States OA is the most common joint disorder in the world, but there is no approved therapeutics to prevent disease progression Historically, OA has been considered a wear-and-tear joint disease, and efforts to identify and develop disease-modifying therapeutics have been predominantly focused on direct inhibition of cartilage degeneration However, now there is increasing evidence that inflammation is a key mediator of OA joint pathology, and also the link between obesity and that OA is not solely due to excessive load-bearing, therefore suggesting that targeting inflammation in OA could be a rewarding therapeutic strategy[3] The proinflammatory cytokines involved in OA, TNF-α and IL1β, are considered the major implicated[3] TNF-α promotes inflammation in blood vessel walls to induce endothelial cell ACCEPTED MANUSCRIPT injury, and regulate leukocyte activation, maturation and release of cytokines and chemokines[4] Cartilage degradation is a central event of OA, driven by an imbalance of metabolic signals and perpetuated by degradative metallo-proteinases (MMP)[5] MMPs RI PT are categorized into the following groups: collagenases (MMP-1, MMP-8, and MMP-13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-10, and MMP-11), matrilysin (MMP-7), metalloelastase (MMP-12), and membrane-typematrix metalloproteinases (MT-MMP-1, 2, 3, and 4)[6] A direct route into the bloodstream via the subchondral microcirculatory system and an indirect route from synovial fluid into SC circulation could explain the higher plasma levels of MMP-9 in OA OA with symptoms of mild to moderate pain, the first line of therapy to cure this condition M AN U is the use of nonsteroidal anti-inflammatory drugs (NSAID) However, the side effects of gastrointestinal and cardiovascular risk of NSAIDs limited long-term use of NSAIDs specifically in geriatric patients Special attention needs to be recommended when NSAIDs are administered to patients with cardiovascular risk, as well as the use of selective COX-2 inhibitors[7] Other therapies commonly used are supplements containing glucosamine and chondroitin sulfate[8] The combination of these supplements are derived from the processing of marine animals, consequently, glucosamine and chondroitin sulfate cannot be consumed TE D by patients with OA who have a history of allergy to marine animals[9] Because the drugs to treat OA are still limited, this becomes one attention of researchers to find potential drug candidates that can cure OA from natural products Babandotan leaves [Ageratum conyzoides L (A conyzoides L)] is natural product that has EP anti-inflamasi effect[10] Previous research showed anti-inflammatory activity of hydroalcoholic babandotan leaves extract at the dose of 250-500 mg/kg in rat model of AC C chronic inflammation[11] The anti-inflammatory activity of babandotan leaves by inhibition of IL-6 Flavonoid glycoside compounds in the extracts of A conyzoides leaves has an important role as an anti-inflammatory compound[12] In this study, the effects of A conyzoides L on the monosodium iodoacetate induced OA rats were analyzed to evaluate its effects on TNF-α and MMP-9 which control inflammation and degradation of proteoglycan on the OA Material and methods ACCEPTED MANUSCRIPT 2.1 Chemicals and reagents Monosodium iodoacetate, ethanol 96%, methanol p.a, ethyl acetate p.a and petroleum ether p.a were obtained from Sigma (St Louis, MO, USA) TNF-α ELISA kit (Catalogue No.: RI PT ER1393) and MMP-9 ELISA Kit (Catalogue No.: ER139) were purchased from Finetest® All other reagents were analytical grade 2.2 Plant materials SC A conyzoides (babandotan) leaves as the main material, was obtained from the Research Institute for Spices and Medicinal Plants, Bogor and determined by the Research Center for Indonesian Institute of Sciences (Certificate of Determination No M AN U Biology, 206/IPH.1.01/If.07/I/2016) This plant was harvested from plants grown in Bogor, Indonesia (Bogor climate: temperature 21.8-30.4 ℃/average 26 ℃, humidity 70%, rainfall is quite high average 500 to 000 mm per year, altitude 190-133 m above sea) 2.3 Preparation of babandotan leaves extract TE D The dried powder of babandotan leaves (A conyzoides L.) were extracted by maceration method using ethanol Then the extract was concentrated by rotary evaporator (Eyela, Tokyo Rikakikai, Tokyo, Japan) The extractive value of ethanol from dried powder was calculated EP as % w/w yield and was found to be 16.2 % AC C 2.4 Identification and quantification of quercetin content of babandotan A total of 20 mL sample solution (10 mg/mL) was spotted on a silica gel 60 F254 plate using automatic thin layer chromatography (TLC) sampler The elution process was performed until the eluent reaches the finish line Eluent composisition was toluene: ethyl acetate: format acid (5:4:0.2) The spot then was evaluated by automatic TLC sampler and TLC equipment identity (CAMAG, Switzerland) 2.5 Animals ACCEPTED MANUSCRIPT White male Sprague-Dawley rats, aged 30 d, were purchased from Indonesia National Institute of Health Research and Development The animals were grouped and housed in polyacrylic cages and maintained under standard laboratory conditions [temperature (25 ± 2) ℃] with dark and lightcycle (12/12 h) and allowed free access to commercial pellet dietand RI PT water ad libitum This research had been certified by ethical certification of Faculty of Medicine, University of Indonesia (UI FK No 75/UN2.F1/Ethics/2016) for the use of animals in experiments These Sprague-Dawley rats were divided into groups: normal control and negative control groups, both given 0.5% carboxymethyl cellulose; the positive control group, given SC glucosamine and chondroitin suspension (486 mg/200 g B.W.); and dose variation extract groups including dose 1, and that were given 40, 80, and 160 mg/200 g B.W respectively M AN U on day 29 until 50 All the groups were induced with 0.05 mL monosodium iodoacetate (20 mg/mL) on day 1, except normal control induced by saline Rats were induced by 50 µL monosodium iodoacetate (20 mg/mL) by intraarticular injection; furthermore, three doses of extracts of babandotan leaves were administrated on day 29 until day 49 after monosodium iodoacetate injection The edema was evaluated every week TE D 2.6 Hematology analysis Hematology data were taken on day 0, 29 and 50 and blood were collected from orbital EP sinus and analyzed by Haematology Analyser (Medonic, Sweden) AC C 2.7 Detection of TNF-α and MMP-9 levels Blood samples were collected at day 50 and then centrifuged at 000 rpm for 15 to get serum and keep in -30 ℃ until analyzed TNF-α and MMP-9 were determined by ELISA according to the manufacturer’s instructions, and read the absorbance using microplate reader (Biochrom, Holliston, USA) 2.8 Histopathological investigation of knee joint Histology data was conducted after sacrificed the rats and isolated the knee joint After kinds of histology procedure to prepare isolated knee joint to become slice of knee joint, then ACCEPTED MANUSCRIPT the slices were stainned by hematoxyllin and eosin (H&E) and safranin O- fastgreen The histological changes were observed under laser scanning microscopy (Olympus, Tokyo, Japan) RI PT 2.9 Statistical analysis Statistics software (SPSS version 16.0) was used for statistical analysis The data represented mean ± standard error of the mean Statistical calculations were analyzed by Oneway ANOVA followed by multiple comparison tests P value